Fabrication of a fluorescent material for sensing an analyte

ABSTRACT

An analyte indicator may include a porous base and may be included in an analyte sensor. The analyte indicator may retain its physical, chemical, and optical properties in the presence of compression. The porous base may not vary in opacity. The analyte indicator may include (i) a polymer unit attached or polymerized onto or out of the porous base and (ii) an analyte sensing element attached to the polymer unit or copolymerized with the polymer unit. The analyte sensing element may include one or more indicator molecule. The analyte sensing element may include one or more indicator polymer chains. The analyte indicator may include (i) an indicator polymer chain attached or polymerized onto or out of the porous base and (ii) indicator molecules attached to the indicator polymer chain.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is continuation of U.S. patent application Ser.No. 15/606,260, which was filed on May 26, 2017, and is a divisional ofU.S. patent application Ser. No. 14/807,033, which was filed on Jul. 23,2015, now U.S. Pat. No. 9,778,190, and claims the benefit of priority toU.S. Provisional Application Ser. No. 62/027,997, which was filed onJul. 23, 2014, all of which are incorporated herein by reference intheir entireties.

BACKGROUND Field of Invention

The present invention relates to analyte indicators. Specifically, thepresent invention relates to an analyte indicator including an analytesensing element attached to or copolymerized with a polymer unit orchain that is attached or polymerized onto or out of a porous base.

Discussion of the Background

Existing analyte indicators may vary in physical, chemical, and/oroptical properties in the presence of compression, and the variation mayreduce the sensitivity of sensors incorporating the existing analyteindicators. Moreover, existing analyte indicators may require a longperiod of time to hydrate. Accordingly, there is a need for an improvedanalyte indicator.

SUMMARY

One aspect of the invention may provide an analyte indicator, which maybe included in an analyte sensor. The analyte indicator may include aporous base, a polymer unit, and an analyte sensing element. The polymerunit may be attached or polymerized onto or out of the porous base. Theanalyte sensing element may be attached to the polymer unit orcopolymerized with the polymer unit.

In some embodiments, the porous base may not vary in opacity. In someembodiments, the analyte indicator may retain its physical, chemical,and optical properties in the presence of compression (e.g., from amembrane or other source). In some embodiments, the polymer unit may behydrophilic or amphiphilic.

In some embodiments, the analyte sensing element may be one or moreindicator molecule. In some embodiments, the analyte sensing element mayinclude an indicator polymer chain. In some embodiments, the indicatorpolymer chain may include indicator molecules. In some embodiments, theanalyte sensing element may include indicator polymer chains, and eachof the indicator polymer chains of the analyte sensing element mayinclude indicator molecules. In some embodiments, the indicator polymerchain may be hydrophilic or amphiphilic.

In some embodiments, the analyte indicator may include a second polymerunit and a second analyte sensing element. The second polymer unit maybe attached or polymerized onto or out of the porous base. The secondanalyte sensing element may be attached to the second polymer unit orcopolymerized with the second polymer unit. In other embodiments, theanalyte indicator may include a third or more polymer units and a thirdor more analyte sensing elements.

Another aspect of the invention may provide an analyte indicator, whichmay be included in an analyte sensor. The analyte indicator may includea porous base, an indicator polymer chain, and indicator molecules. Theindicator polymer chain may be attached or polymerized onto or out ofthe porous base. The indicator molecules may be attached to theindicator polymer chain.

In some embodiments, the porous base may not vary in opacity. In someembodiments, the analyte indicator may retain its physical, chemical,and optical properties in the presence of compression (e.g., from amembrane or other source). In some embodiments, the polymer unit may behydrophilic or amphiphilic.

In some embodiments, a porous membrane may be wrapped tightly over theanalyte indicator.

Further variations encompassed within the systems and methods aredescribed in the detailed description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various, non-limiting embodiments ofthe present invention. In the drawings, like reference numbers indicateidentical or functionally similar elements.

FIG. 1A is a macroscale interpretation of an analyte indicator embodyingaspects of the present invention in which polymer units are attached tothe porous base and then modified to contain either an indicatormolecule or one or more indicator polymer chains yielding a branchedsystem.

FIG. 1B is a blown-up view of a polymer unit containing indicatorpolymer chains embodying aspects of the present invention.

FIG. 2A illustrates an analyte indicator embodying aspects of thepresent invention in which one or more indicator polymer chains areattached or polymerized onto or out of a base membrane layer.

FIG. 2B shows the base monomer make-up of a grafted linear copolymer inwhich R₁, R₂, and R₃ are hydrophilic acrylate-based monomers such as butnot limited to 2-hydroxyethyl methacrylate (HEMA), poly(ethylene glycol)methacrylate (PEGMA), and/or acrylic/methacrylic acid embodying aspectsof the present invention.

FIGS. 3A and 3B are graphs showing the emission light intensity andresponse time, respectively, of an analyte indicator embodying aspectsof the present invention.

FIGS. 4A and 4B are graphs comparing glucose response curves of ahydrogel analyte indicator with the response time of an analyteindicator embodying aspects of the present invention, respectively.

FIG. 5 is graph showing signal performance of an analyte indicatorembodying aspects of the present invention in the presence of 30 mg/mlprotein.

FIG. 6 is graph comparing of an analyte indicator embodying aspects ofthe present invention in the presence of protein to a hydrogel analyteindicator as a function of high (70 mg/mL) protein fouling.

FIG. 7 is a graph showing the time required for post-sterilizationhydration of an analyte sensing substrate embodying aspects of thepresent invention in saline.

FIG. 8 is a graph showing post-ethylene oxide (ETO) sterilizationglucose responsivity of an analyte indicator embodying aspects of thepresent invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Analyte systems in accordance with embodiments of the present inventionmay include an analyte sensor and a transceiver. In some embodiments,the analyte system may be a continuous analyte monitoring system. Insome non-limiting embodiments, the system may be 24-hour continuousanalyte monitoring system configured to measure analyte levels. In onenon-limiting embodiment, the continuous analyte monitoring system may bea continuous glucose monitoring system (CGMS) and may measureinterstitial fluid glucose levels in adults who have diabetes mellitus.

In some embodiments, the analyte sensor may be a subcutaneouslyimplantable sensor (e.g., in the back of the upper arm, wrist, or lowerabdomen) with no sensor part protruding from the skin, and thetransceiver may be a battery-powered transceiver configured towirelessly receive data from the analyte sensor (e.g., via inductivecommunication) and function as a data collection and monitoring device.However, this is not required, and, in alternative embodiments, theanalyte sensor may be a transcutaneous sensor having a wired connectionto the transceiver. In some embodiments, the transceiver sends data to asmartphone (or other device, such as a receiver, laptop, or personalcomputer). The smartphone may execute a medical application (e.g., amobile medical application).

In some embodiments, the analyte sensor may be encased in abiocompatible material. In some embodiments, the sensor utilizes afluorescent, analyte-indicating macromolecule. In some embodiments, thesensor may last up to 6 months and may be replaced thereafter. In someembodiments, the sensor may include a light source (e.g., light emittingdiode). The sensor may include a polymer. The light source may excitethe polymer, and the polymer may rapidly signal changes in analyteconcentration via a change in light output, which may be measured by thesensor (e.g., by a photodetector of the sensor). In some embodiments,the sensor relays the measurement to the transceiver. In someembodiments, the entire measurement may be done autonomously andindependently without any prompting by the user.

In some embodiments, the fluorescent analyte chemistry is not subject toinstabilities intrinsic to current protein-based analyte sensors. Forinstance, in some embodiments, the analyte measurement by the sensorneither consumes chemicals (e.g., oxygen, glucose) nor forms chemicals(e.g., hydrogen peroxide). Consequently, sensors in accordance withembodiments of the invention may be inherently more stable and accurate,and an implanted sensor may last for up to 6 months or longer beforebeing replaced.

In some embodiments, the analyte-indicating polymer of the analytesensor may include indicator molecules, such as, for example and withoutlimitation, any of the indicator molecules described in U.S. PatentApplication Publication No. 2014/0088383, which is incorporated byreference in its entirety. In some non-limiting embodiments where theanalyte monitoring system is a glucose monitoring system, the indicatormolecules may be fluorescent indicator molecules that reversibly bindglucose. In one non-limiting embodiment, when a fluorescent indicatormolecule has bound glucose, then the indicator molecule becomesfluorescent, in which case it absorbs (or is excited by) light at awavelength of approximately 378 nm and emits light in the range of 400to 500 nm. When no glucose is bound, then the indicator molecule may beonly weakly fluorescent. In a non-limiting embodiment, the indicatormolecules may be embedded in a polymer graft that covers only a smallportion of the sensor.

In some existing “hydrogel” embodiments, the polymer graft may be a“hydrogel” containing three monomers: (i) the TFM fluorescent indicator,(ii) hydroxyethylmethacrylate (HEMA), which is a methacrylate, and (iii)polyethylene glycol diacrylate (PEG-diacrylate). The three monomers maybe in specific molar ratios. For example, in one hydrogel embodiment,the fluorescent indicator may comprise 0.1 molar percent, HEMA maycomprise 94.3 molar percent, and PEG may comprise 5.6 molar percent. ThePEG may act as a cross-linker and create a sponge-like matrix/gel. ThePEG may increase hydrophilicity. In some embodiments, the hydrogelpolymer graft may be synthesized using conventional free radicalpolymerization techniques.

In these existing hydrogel embodiments, the hydrogel polymer graft maybe a sponge-like substance and, therefore, sensitive to mechanicaldamage and opacity changes. If the hydrogel polymer graft is putdirectly in contact with body tissues, the indicator component may berapidly oxidized, which limits the lifetime of the analyte sensor. Inaddition, if a membrane (e.g., a nylon membrane) is placed over thehydrogel graft (i.e., between the hydrogel graft and the body tissues),the membrane may compress the hydrogel graft, and, as a result, thehydrogel may be incapable of expanding to its original state. Thecompression by the membrane placed over the hydrogel may change thehydrogel's opacity or ability to scatter the fluorescent signalefficiently back to photodetector(s) (e.g., photodiode(s)) of theanalyte sensor, reducing the sensor's sensitivity and optical stability.Therefore, if the membrane fits too snugly around the hydrogel, thesensor's sensitivity may be reduced. Additionally, if the membrane isnot snug, then the hydrogel may still degrade, or the analyte (e.g.,glucose) may take too long to diffuse into the hydrogel, making thesensor less responsive. Furthermore, bulk hydrogel may require days tofully hydrate, which imposes an undesired wait time limitation on theproduct use. Moreover, the bulk hydrogel is opaque (white), and thatopacity can be affected by solute (e.g., by the protein concentration inthe solute), which may affect the in vivo analyte accuracy of thesensor. Further, when the hydrogel is compressed, it cannot fullyhydrate to its whitest state and will remain translucent or opalescentbased on how much compression is placed onto the hydrogel, and thiswould add a significant amount of error into the analyte measurement.

Some embodiments of the present invention may provide an analyteindicator to replace the hydrogel system and address one or more issues(e.g., variation in physical, chemical, and/or optical properties of thehydrogel in the presence of compression and/or variation in the opacityof the hydrogel (white to clear) that can change the hydrogel's abilityto scatter the signal back into the sensor). Moreover, in someembodiments, the analyte indicator may have a short hydration time(e.g., minutes) as opposed to the hours to days required for thehydrogel.

FIG. 1A illustrates an analyte indicator 100 embodying aspects of thepresent invention. In some embodiments, the analyte indicator 100 mayinclude a porous base membrane layer 101. In some non-limitingembodiments, the porous base 101 may be comprised of nylon (e.g., Nylon6, 6). However, this is not required, and, in some alternativeembodiments, the porous base 101 may be comprised of other, similarmembrane materials, such as, for example and without limitation,cellulose acetate, polypropylene, polyether sulfone, polyethylene,polyvinylidene difluoride (PVDF), polycarbonate, polytetrafluoroethylene(PTFE), or polyethylene terephthalate (PET). In some non-limitingembodiments, the base membrane layer 101 does not vary in opacity. Insome non-limiting embodiments, the porous base 101 may retain itsphysical, chemical, and optical properties in the presence ofcompression. As illustrated in FIG. 1A, in some embodiments, the basemembrane layer 101 may include long, connected strands.

In some embodiments, as illustrated in FIG. 1A, the analyte indicator100 may include a polymer 102 attached or polymerized onto or out of theporous base 101. In some embodiments, the polymer 102 may be in units(e.g., strands) that are attached to or polymerized off of the backboneprovided by the base membrane layer 101. In some non-limitingembodiments, the polymer 102 may be polyethylene glycol (PEG). However,this is not required, and, in alternative embodiments, other materialsmay be used, such as, for example and without limitation,poly(oxazolines), poly(acrylamides), poly(electrolytes), poly(ethers),poly(vinyl pyrolidone), Poly(ethylenimines), poly(vinyl alcohol),poly(acrylates and methacrylates), and/or poly(maleic anhydride). Thepolymer units 102 may provide a flexible structure that retains itsphysical, chemical, and/or optical properties when compressed. In someembodiments, the polymer units may be hydrophilic or amphiphilic. InFIG. 1A, the polymer units 102 are shown as short strands off of thelong strands of the base membrane layer 101.

In some embodiments, the analyte indicator 100 may include one or moreanalyte sensing elements 103. The one or more analyte sensing elements103 may be attached or copolymerized to the polymer units 102. In somenon-limiting embodiments, as illustrated in FIG. 1A, each polymer unit102 may have one analyte sensing element 103 attached or copolymerizedthereto. However, this is not required, and, in some alternativeembodiments, one or more of the polymer units 102 may not have ananalyte sensing element 103 attached or copolymerized thereto. Forexample, in one non-limiting alternative embodiment, a small number ofanalyte sensing elements 103 may be attached to the polymer units 102(e.g., an analyte sensing element 103 may be attached to approximatelyone tenth of the polymer units 102). Moreover, in some alternativeembodiments, one or more of the polymer units 102 may have multiple(e.g., two, three, four or more) analyte sensing elements 103 attachedor copolymerized thereto. In FIG. 1A, the analyte sensing elements 103are shown as circles attached or copolymerized to the polymer units 102.

In some embodiments, one or more of the analyte sensing elements 103 mayconsist of one or more indicator molecules 104 attached to a polymerunit 102. In some embodiments, as illustrated in FIG. 1B, one or more ofthe analyte sensing elements 103 may include one or more indicatorpolymer chains (i.e., linear chains) 105 attached or polymerized onto orout of a polymer unit 102. In this way, the indicator polymer chains 105may branch out from the polymer units 102. Accordingly, in someembodiments, the analyte indicator 100 may have a branched polymerstructure. The indicator polymer chains 105 may include one or moreindicator molecules 104 attached thereto. Although FIG. 1B illustratesan analyte sensing element 103 having three indicator polymer chains 105attached or polymerized onto or out of a polymer unit 102, this is notrequired, and, in some alternative embodiments, an analyte sensingelement 103 may have a different number (e.g., one, two, four, five,etc.) of indicator polymer chains 105 attached or polymerized onto orout of a polymer unit 102. In some embodiments, although not illustratedin FIG. 1B, one or more of indicator polymer chains 105 may have one ormore indicator polymer chains 105 attached or polymerized onto or out ofthe indicator polymer chain 105 for additional branching.

In some non-limiting embodiments, the indicator polymer chains 105 maybe short (e.g., 1-200 nm). In some embodiments, the overall structure ofthe analyte indicator 100 including the one or more indicator polymerchains 105 retains its physical, chemical, and/or optical properties inthe presence of compression from an external source (e.g., a secondarymembrane wrapped on top of the analyte indicator). In some embodiments,the polymer chains 105 could consist of, for example and withoutlimitation, 2-hydroxyethylmethacrylate, poly(ethylene glycol)methacrylate, acrylic acid, methacrylic acid,[2-(methacrylolyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide,or vinyl pyrrolidone. However, in some alternative embodiments, othermaterials may be used for the polymer chains. In some embodiments, theindicator polymer chains 105 may be hydrophilic or amphiphilic.

In some non-limiting embodiments, the analyte indicator 100 may beformed by making the polymer chain(s) 105 with the indicator molecules104 attached thereto and then attaching polymer chain(s) 105 to thepolymer unit(s) 102, which may be already be attached or polymerizedonto or out of the porous base 101. However, this is not required, and,in alternative embodiments, the analyte indicator 100 may be formed indifferent manners.

FIG. 2A illustrates an analyte indicator 200 embodying aspects of thepresent invention. Similar to the analyte indicator 100 illustrated inFIG. 1A, the analyte indicator 200 may include a porous base membranelayer 101. The analyte indicator 200 may include one or more indicatorpolymer chains 105 attached or polymerized onto or out of the basemembrane layer 101. Accordingly, the analyte indicator 200 may have alinear polymer structure.

In some embodiments, as illustrated in FIG. 2A, the linear polymerchains 105 may be grafted onto a surface of the base membrane layer 101.FIG. 2B shows the base monomer make-up of a grafted linear copolymer105. In some embodiments, R₁, R₂, and R₃ may be hydrophilicacrylate-based monomers such as but not limited to 2-hydroxyethylmethacrylate (HEMA), poly(ethylene glycol) methacrylate (PEGMA), and/oracrylic/methacrylic acid. In some non-limiting embodiments, theindicator polymer chains 105 of the analyte indicator 100 illustrated inFIG. 1B may have the base monomer make-up illustrated in FIG. 2B.

In some non-limiting embodiments, the analyte indicator 200 may beformed by making the polymer chain(s) 105 with the indicator molecules104 attached thereto and then attaching polymer chain(s) 105 to theporous base 101. However, this is not required, and, in alternativeembodiments, the analyte indicator 200 may be formed in a differentmanner.

In some embodiments, the indicator material (e.g., fluorescent indicatormaterial) is hydrophilic, amphiphilic, and/or optically stable. In someembodiments, the analyte indicator (e.g., analyte indicator 100, whichmay have a branched polymer structure, or analyte indicator 200, whichmay be a linear copolymer graft membrane) may be wrapped tightly withina secondary membrane-based system without variance of the physical,chemical, and optical properties of the analyte indicator in thepresence of substantial compression of the analyte indicator by thesecondary membrane-based system. In some non-limiting embodiments,additional materials could also be tightly wrapped around the analyteindicator without interfering with the analyte indicator's properties.The additional materials may provide different layers, such as, forexample, a layer of porous material to protect the analyte indicatorfrom degradation and/or another layer of a dark material to make thesensor less sensitive to ambient light. In some embodiments, the analyte(e.g., glucose) may flow freely through the material, as the materialmay be hydrophilic and porous.

In some embodiments, the analyte indicator may be attached to theanalyte sensor by O₂ plasma treating the sensor followed by tack weldingthe analyte indicator to the sensor at 450° F. (230° C.). However, thisis not required, and, in alternative embodiments, the analyte indicatormay be attached to the analyte sensor using a different method. In someembodiments, analyte indicator is attached to the sensor in a mannerthat allows intimate contact of the analyte indicator (e.g., analyteindicator 100, which may have the branched polymer structure, or analyteindicator 200, which may be a linear copolymer graft membrane) with theencasement (e.g., the PMMA encasement) of the sensor platform (e.g., bycutting the analyte indicator to 0.18″×0.47″ when used with a sensorundercut width of 0.193″).

In some embodiments, the analyte indicator (e.g., analyte indicator 100,which may have the branched polymer structure, or analyte indicator 200,which may be a linear copolymer graft membrane) has one or more of thefollowing advantages: (i) ability to be produced on a large scale andstored, (ii) elimination of hydration before implant (i.e., allows fordry implant), (iii) retention of its physical, chemical, and opticalproperties in the presence of compression, (iv) optical stability, (v)built-in oxidative stability, (vi) fast response times, and (vii) atuneable K_(d).

In one non-limiting embodiment, the analyte indicator showed amodulation (e.g., 80-190%) equivalent to and greater than the modulationof the hydrogel system (e.g., 70-90%) as well as good to excellent T₉₀response time data (e.g., 3.5 to 5 min) compared with the T₉₀ responsetime of the hydrogel system having nylon (e.g., 8.6±1.0 min). See FIGS.3A, 3B, 4A, and 4B.

In one non-limiting embodiment, the analyte indicator may have a Tauvalue of 7±2 minutes. In one non-limiting embodiment, the analyteindicator has an average Tau of 9 minutes (9.5 minutes with PET)compared with an average Tau value of 17 minutes for the hydrogelwrapped with nylon. See FIG. 4.

In some embodiments, the analyte indicator may have tunable Kd values.For example, in non-limiting embodiment, an analyte indicatorformulation may have a targeted K_(d) of 15 and an actual K_(d) of13.7±0.4, a targeted K_(d) of 18 and an actual K_(d) of 17±1.3, atargeted K_(d) of 20 and an actual Kd of 21, or a targeted Kd of 22 andan actual K_(d) of 23±0.7.

In some embodiments, absolute modulation may be on the same scale as thehydrogel chemistry but with a lower S₀ allowing for the sensitivity ofthe gain to be adjusted to be less sensitive towards ambient light.

In some embodiments, the sensor having the analyte indicator (e.g.,analyte indicator 100 or 200) shows no drop in signal/sensor performancein the presence of bovine serum albumin (BSA) at a concentration of 30mg/mL. See FIG. 5. FIG. 6 is a chart showing experimental resultscomparing one embodiment of the analyte indicator to the hydrogel systemas a function of protein fouling at 70 mg/mL.

In some embodiments, rehydration of the analyte indicator (e.g., thelinear copolymer graft membrane system) occurs quickly (e.g., in onenon-limiting embodiment, the analyte sensor may take less than 5 minutesto reach a stable signal) compared to the nylon-wrapped hydrogel system,which requires 2-2.5 hours of rehydration for the nylon-wrapped hydrogelto reach a stable signal.

In some non-limiting embodiments, the analyte indicator is sterilized(e.g., using ethylene oxide (ETO)), and a stable signal may be reachedafter approximately four minutes of post-sterilization hydration bydipping the sensor in saline solution. See FIG. 7. In some non-limitingembodiments, the analyte indicator (e.g., the linear copolymer graftmembrane system) may have a T₉₀ response time of 4.8±0.3 minutespost-ETO sterilization. See FIG. 8.

Embodiments of the present invention have been fully described abovewith reference to the drawing figures. Although the invention has beendescribed based upon these preferred embodiments, it would be apparentto those of skill in the art that certain modifications, variations, andalternative constructions could be made to the described embodimentswithin the spirit and scope of the invention.

What is claimed is:
 1. An analyte indicator comprising: a porous base; apolymer unit polymerized onto or out of the porous base, wherein thepolymer unit is a polyethylene glycol (PEG) unit; and an analyte sensingelement attached to the polymer unit or copolymerized with the polymerunit, wherein the analyte sensing element includes an indicator polymerchain attached or polymerized onto or out of the polymer unit andindicator molecules attached to the indicator polymer chain, theindicator molecules neither consume glucose nor form hydrogen peroxide,and the indicator polymer chain consists of 2-hydroxyethylmethacrylate,poly(ethylene glycol) methacrylate, acrylic acid, methacrylic acid,[2-(methacrylolyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide,or vinyl pyrrolidone.
 2. The analyte indicator of claim 1, wherein theanalyte indicator retains its physical, chemical, and optical propertiesin the presence of compression.
 3. The analyte indicator of claim 1,wherein the porous base does not vary in opacity.
 4. The analyteindicator of claim 1, wherein the polymer unit is hydrophilic oramphiphilic.
 5. The analyte indicator of claim 1, wherein the analytesensing element comprises indicator polymer chains, and each of theindicator polymer chains of the analyte sensing element comprisesindicator molecules.
 6. The analyte indicator of claim 1, wherein theindicator polymer chain is hydrophilic or amphiphilic.
 7. The analyteindicator of claim 1, wherein the base comprises nylon, cellulose,cellulose acetate, polypropylene, polyethylene, poly(ethyleneterephthalate), poly(ether sulfone), poly(vinylidene difluoride), orpoly(tetrafluoroethylene).
 8. The analyte indicator of claim 1, whereinthe porous base is flexible.
 9. The analyte indicator of claim 1,further comprising: a second polymer unit attached or polymerized ontoor out of the porous base; and a second analyte sensing element attachedto the second polymer unit or copolymerized with the second polymerunit, wherein the second analyte sensing element includes a secondindicator polymer chain and second indicator molecules attached to thesecond indicator polymer chain.